GAMMA-2 CHAIN OF LAMININ-5 IS RECOGNIZED BY MONOCLONAL-ANTIBODY GB3

Herlitz junctional epidermolysis bullosa is an autosomal recessive dis order characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane, The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patien t skin, The antigen recognized by GB3 has been identified as laminin-5 , a glycoprotein consisting of three subunits (alpha 3, beta 3 and gam ma 2). To identify the laminin-5 protein chain that contains the epito pe recognized by GB3 and to determine if chain assembly is required fo r antibody recognition, we expressed a gamma 2 protein constructed fro m a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclon al antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion exper iments, each antibody removed most of the protein that was reactive wi th the other antibody, The epitope recognized by GB3 is present only w hen the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immu noblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells ( gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluoresence staining, this epito pe may be dependent upon a less stable conformation of gamma 2. We con clude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form ofthe gamma 2 chain.